The long-term goal of this project is to define bacterial virulence factors and host responses that determine the outcome of serious, disseminated Salmonella infections. Non-typhoid Salmonella strains are common causes of food-borne illness in the United States and have been used effectively in a bioterrorism attack in this country. In addition, Salmonella represent an important research tool to study the molecular biology and immunology of the host-pathogen interaction. This project focuses on the Salmonella spv locus, a critical virulence determinant required for systemic disease. The spv operon encodes SpvB, an ADP-ribosyl transferase that modifies actin monomers in infected cells and leads to loss of the actin cytoskeleton. The active NAD-binding site of SpvB is located in the C-terminal domain and is required for the virulence phenotype. SpvB is the first ADP-ribosylating toxin that acts specifically from bacteria located intracellularly, and the first actin modifying toxin shown to be crucial for intracellular pathogenesis. This project will focus on the novel aspects of SpvB secretion and transport that allow the toxin to access the host cell from the phagosome rather than from the extracellular environment. The mechanisms of cytotoxicity and cell death mediated by SpvB will be determined. The Specific Aims are: 1) to analyze the mechanism of SpvB secretion and transport from the phagosome into the host cell cytoplasm. The hypothesis is that this process requires both the SPI2-encoded bacterial type III secretion system and specific regions of the SpvB protein. 2) to define the role and function of the N-terminal domain of SpvB in intracellular infection. The hypothesis is that the N-terminal domain contains regions that specify transport from the phagosome into the host-cell cytoplasm. 3) to determine the pathophysiologic consequences of actin depolymerization in the host cell during intracellular infection by Salmonella. The hypothesis is that actin depolymerization disrupts multiple aspects of cellular function, enhances intracellular growth, and triggers a delayed cell death pathway that enhances the cell-to cell spread of Salmonella during the infectious process. [unreadable] [unreadable]